Neuronal excitability is mediated by ion-specific channels which allow specific ions to cross cell membranes to generate action potentials. Voltage dependent sodium channels are responsible for the initial rising phase of the action potential (Nicholls et al., 1992, Ionic Basis of the Action Potential, pp. 90-120, edited by J. J. Nicholls, Sinauer Associates, Inc., Suderland, Mass.). In vertebrates, sodium channels in the brain, muscle and other tissues are large membrane glycoprotein complexes composed of an .alpha. subunit (230-270 kDa) and 1-2 tightly associated smaller (33-38 kDa) .beta. subunits (reviewed by Catterall, 1992, Physiol. Rev., vol. 72, S2-S47). The large .alpha. subunit forms the ion permeable pore while the smaller subunits play key roles in the regulation of channel function (Isom et al., 1992, Science, vol. 256, 839-842; reviewed by Isom et al., 1994, Neuron, vol. 12, 1183-1194). The structure of invertebrate sodium channels has not been well defined. However, gene cloning studies establish the existance of .alpha.-subunits of structure similar to those described for vertebrates (Loughney et al., 1989, Cell, vol. 58, 1143-1154; Ramaswami and Tanouye, 1989, Proc. Natl. Acad. Sci. USA, vol. 86, 2079-2082; Okamoto et al., 1987, Proc. Jpn. Acad., vol. 63, 284-288).
Analysis of behavioral mutants provides a unique genetic approach to dissect the molecular components underlying neuronal membrane excitability without requiring any a priori information of the gene product (See reviews by Hall, 1982, Quart. Rev. Biophys., vol. 15, 223-479; Ganetzky and Wu, 1986, Annu. Rev. Genet., vol. 20, 13-44; Salkoff and Tanouye, 1986, Physiol. Rev., vol. 66, 301-329; Tanouye et al., 1986, Annu. Rev. Neurosci., vol. 9, 255-276; Papazian et al., 1988, Ann. Rev. Physiol., vol. 50, 379-394; Wu and Ganetzky, 1992, Neurogenetic Studies of Ion Channels in Drosophila, pp. 261-314 in Ion Channels, Vol. 3, edited by T. Narahashi, Plenum Press, New York). Therefore, this approach has the potential to identify new gene products which would not be isolated by biochemical methods or homology cloning.
One particular group of behavioral mutants, including para (paralytic, Suzuki et al., 1971, Proc. Nat. Acad. Sci. USA, vol. 68, 890-893), nap (no action potential, Wu et al., 1978, Proc. Natl. Acad. Sci. USA, vol. 75, 4047-4051), tipE (temperature-induced paralysis, locus E, Kulkarni and Padhye, 1982, Genet. Res., vol. 40, 191-199), and sei (seizure, Jackson, et al., 1984, Nature, vol. 308, 189-191; Jackson et al., 1985, J. Neurosci., vol. 5, 1144-1151), originally isolated by their phenotype of temperature-sensitive paralysis, has been proposed to affect sodium channels in Drosophila. For example, ligand binding studies with sodium channel-specific neurotoxins showed that head membranes from nap and tipE had a decreased number of saxitoxin binding sites, while different sei alleles affected the number or the affinity of saxitoxin binding sites (Jackson et al., 1984, cited elsewhere herein; Jackson et al., 1986, J. Neurogenet., vol. 3, 1-17). Whole cell patch clamp studies showed that cultured embryonic neurons from sei and tipE have reduced sodium currents (O'Dowd and Aldrich, 1988, J. Neurosci., vol. 8, 3633-3643), while para alleles have a decrease in the fraction of neurons which express sodium currents (O'Dowd et al., 1989, Neuron, vol. 2, 1301-1311). Molecular cloning of para revealed that it encodes a Drosophila sodium channel .alpha. subunit (Loughney et al., 1989, Cell, vol. 58, 1143-1154) while nap is a DNA binding protein which may regulate para expression by binding to the X chromosome where para is located (Kernan et al., 1991, Cell, vol. 66, 949-959).
The tipE mutation is an ethyl methane sulfonate-induced recessive mutation. Homozygous tipE flies paralyze rapidly at 38.degree. and recover immediately when returned to 23.degree. (Kulkarni and Padhye, 1982, cited elsewhere herein). Besides the results from ligand binding and electrophysiological studies discussed above, double mutant studies of tipE with para and nap provided additional evidence that tipE affects sodium channels. The combination of tipE with nap or tipE with various para alleles resulted in unconditional lethality of the double mutants at temperatures where single mutants survive (Jackson et al., 1986, cited elsewhere herein; Ganetzky, 1986, J. Neurogenet., vol. 3, 19-31). Interestingly, the synergistic interaction of tipE and para is allele-dependent. The combination of tipE with some para alleles allows varying degree of viability while with other alleles results in complete lethality. The observation that the allele-dependence is not correlated to the residual para sodium channel activities of the different alleles led to the speculation that tipE gene product may physically interact with para (Jackson et al., 1986, cited elsewhere herein; Ganetzky, 1986, cited elsewhere herein). Surviving double mutants of tipE with either para and nap are very weak, and exhibit enhanced temperature sensitivity for paralysis (Jackson et al., 1986, cited elsewhere herein; Ganetzky, 1986, cited elsewhere herein). The tipE and nap double mutants also displayed a greater reduction in saxitoxin binding activity than either single mutant homozygote (Jackson et al., 1986, cited elsewhere herein).
Although some types of sodium channel .alpha.-subunits alone are sufficient to form functional channels when expressed in Xenopus oocytes, their properties are not normal. Inactivation is slower and voltage dependence is shifted to more positive membrane potentials compared to channels in intact neurons. Coexpression of .alpha.-subunits with low molecular weight RNA from rat brain (presumably containing .beta.1 and .beta.2 subunits) not only corrected the abnormality but also dramatically increased the level of expressed sodium current (Auld et al., 1988,, Neuron, vol. 1, 449-461; Krafte et al, 1988, J. Neurosci., vol. 8, 2859-2868; Krafte et al., 1990, J. Gen. Physiol., vol. 96, 689-706). Similar results were obtained when cloned .beta.1 subunit was coexpressed with rat brain .alpha.-subunit (Isom et al., 1992, cited elsewhere herein).
Using a molecular genetic approach, it was determined that the para locus in Drosophila encodes the .alpha. subunit of the voltage dependent sodium channel, and the entire para cDNA sequence was determined (Loughney et al., 1989, cited elsewhere herein; Thackeray and Ganetzky, 1994, J. Neuroscience, vol. 14, 2569-2578). In contrast to some rat brain sodium channel forms, expression of para sodium channel in Xenopus oocytes, or any other expression system, is undetectable. In fact, functional expression of a number of cloned sodium channels in heterologous expression systems has been weak or imposible. These difficult to express channels include those from squid (Rosenthal and Gilly, 1993, Proc. Natl. Acad. Sci. USA, vol. 90, 10026-10030), human heart and uterus (George, et al., 1992, Proc. Natl. Acad. Sci. USA, vol. 89, 4893-4897) and some forms from rat brain (Noda et al., 1986, Nature, vol. 322, 826-828; Noda et al., 1986, Nature, vol. 320, 188-192).
The inability to express insect sodium channel subunits with substantial purity and in an easy assayed system has inhibited the development of rapid and economical assays of sodium channel modulators. Sodium channel modulators have been investigated as insecticides, as therapeutic agents in the treatment and prevention of parasitic infections in humans and domestic animals, and as neuro-protective agents for the treatment of stroke, head injury and other ischemic events. The ability to rapidly screen potential modulators of insect sodium channels would also facilitate the development of compounds for the prevention and treatment of parasitic infections in humans, livestock and domestic animals.
For the foregoing reasons, there remains a need for a method of expressing and isolating a substantially pure form of the voltage dependent cation channel protein.